Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects.

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than ß-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Aminopeptidase A contributes to biochemical, anatomical and cognitive defects in Alzheimer's disease (AD) mouse model and is increased at early stage in sporadic AD brain.

One of the main components of senile plaques in Alzheimer's disease (AD)-affected brain is the Aß peptide species harboring a pyroglutamate at position three pE3-Aß. Several studies indicated that pE3-Aß is toxic, prone to aggregation and serves as a seed of Aß aggregation. The cyclisation of the glutamate residue is produced by glutaminyl cyclase, the pharmacological and genetic reductions of which significantly alleviate AD-related anatomical lesions and cognitive defects in mice models. The cyclisation of the glutamate in position 3 requires prior removal of the Aß N-terminal aspartyl residue to allow subsequent biotransformation. The enzyme responsible for this rate-limiting catalytic step and its relevance as a putative trigger of AD pathology remained yet to be established. Here, we identify aminopeptidase A as the main exopeptidase involved in the N-terminal truncation of Aß and document its key contribution to AD-related anatomical and behavioral defects. First, we show by mass spectrometry that human recombinant aminopeptidase A (APA) truncates synthetic Aß1-40 to yield Aß2-40. We demonstrate that the pharmacological blockade of APA with its selective inhibitor RB150 restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aß-precursor protein (ßAPP). Pharmacological reduction of APA activity and lowering of its expression by shRNA affect pE3-42Aß- and Aß1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA inhibitors and shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of pE3-42Aß-positive plaques, APA activity is augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA is a key enzyme involved in Aß N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with AD pathology.

Contribution of Delta-Opioid Receptors to Pathophysiological Events Explored by Endogenous Enkephalins.

Very few discoveries in the neurosciences have triggered clinical speculation and experimentation regarding the etiology of psychiatric illness to the same extent as that following identification of the opiate receptor(s) and subsequent isolation of endogenous morphine-like peptides. There is overwhelming evidence in animals and in human that opioids are involved in behaviorally relevant issues such as the modulation of pain, the response to stress, motivation, addiction, sexuality, food intake, etc., but our knowledge on the possible relation between opioids and mental illness is still very limited.These responses could be explored eitheir by using higlhy selective delta agonist or by emphasizing the effects of phasically secreted endogenous opioid peptides, enkephalin. Both approaches were investigated in particular through protection of enkephalin degradation by dual enkephalinase ihibitors DENKIs such as RB101, PL37 or PL265.

Substituted α-mercaptoketones, new types of specific neprilysin inhibitors.

New neprilysin inhibitors containing an α-mercaptoketone HSC(R1R2)CO group, as zinc ligand were designed. Two parameters were explored for potency optimization: the size of the inhibitor which could interact with the S1, S1' or S2' domain of the enzyme and the nature of the substituents R1, R2 of the mercaptoketone group. Introduction of a cyclohexyl chain in R1, R2 position and a (3-thiophen)benzyl group in position R3 (compound 12n) yielded to the most potent inhibitor of this series with a Ki value of 2±0.3nM. This result suggests that this new inhibitor interacts within the S1, S1' domain of NEP allowing a pentacoordination of the catalytic Zn2+ ion by the mercaptoketone moiety.

Cocaine dysregulates opioid gating of GABA neurotransmission in the ventral pallidum.

The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a δ and µ opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse.

TGF-ß and opioid receptor signaling crosstalk results in improvement of endogenous and exogenous opioid analgesia under pathological pain conditions.

Transforming growth factor-ß1 (TGF-ß1) protects against neuroinflammatory events underlying neuropathic pain. TGF-ß signaling enhancement is a phenotypic characteristic of mice lacking the TGF-ß pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-ß1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of µ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of µ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-ß1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-ß1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-ß signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity.

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids ß-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [L-(4-benzoyl)phenylalanyl-ß-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k(cat)/K(m)) of 3.8±0.5×104 M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.

Pain inhibition by blocking leukocytic and neuronal opioid peptidases in peripheral inflamed tissue.

Inflammatory pain can be controlled by endogenous opioid peptides. Here we blocked the degradation of opioids in peripheral injured tissue to locally augment this physiological system. In rats with hindpaw inflammation, inhibitors of aminopeptidase N (APN; bestatin) or neutral endopeptidase (NEP; thiorphan), and a dual inhibitor, NH(2)-CH-Ph-P(O)(OH)CH(2)-CH-CH(2)Ph(p-Ph)-CONH-CH-CH(3)-COOH (P8B), were applied to injured paws. Combined bestatin (1.25-5 mg)/thiorphan (0.2-0.8 mg) or P8B (0.0625-1 mg) alone elevated mechanical nociceptive thresholds to 307 and 227% of vehicle-treated controls, respectively. This analgesia was abolished by antibodies to methionine-enkephalin, leucine-enkephalin, and dynorphin A 1-17, by peripherally restricted and by selective µ-, δ-, and κ-opioid receptor antagonists. Flow cytometry and photospectrometry revealed expression and metabolic activity of APN and NEP on macrophages, granulocytes, and sciatic nerves from inflamed tissue. Radioimmunoassays showed that inhibition of leukocytic APN and NEP by bestatin (5-500 µM)/thiorphan (1-100 µM) combinations or by P8B (1-100 µM) prevented the degradation of enkephalins. Blockade of neuronal peptidases by bestatin (0.5-10 mM)/thiorphan (0.1-5 mM) or by P8B (0.1-10 mM) additionally hindered dynorphin A 1-17 catabolism. Thus, leukocytes and peripheral nerves are important sources of APN and NEP in inflamed tissue, and their blockade promotes peripheral opioid analgesia.

Molecular and cellular targets of the MRI contrast agent P947 for atherosclerosis imaging.

P947 (DOTA-Gd-peptide) was recently identified as an MRI contrast agent for the detection and characterization of the matrix metalloproteinases (MMP)-rich atherosclerotic plaques. Because this product displays a broad spectrum affinity for the MMP family, we hypothesized that it may also recognize other metalloproteinases overactivated in vulnerable atherosclerotic plaques. Therefore, this study aimed at describing, at the molecular and cellular level, the interactions between P947 and proteases of atherosclerotic plaques. Fluorimetric assays were used to measure the in vitro affinity of P947 toward recombinant and purified MMPs, angiotensin-converting enzyme (ACE), endothelin-converting enzyme (ECE-1), neutral endopeptidase (NEP), and both aminopeptidases A and N (APA and APN). Using similar fluorimetric assays associated with specific substrates, enzymatic activities were measured in vulnerable and stable plaques collected from human atherosclerotic carotid arteries. Ex vivo affinity of P947 for metalloproteinases in vulnerable lesions was subsequently determined. Interaction between P947 and major cell types present in atherosclerotic plaques was also investigated in different cell lines: PMA-1-differentiated THP-1 (macrophage), Ox-LDL-treated THP-1 (foam cell), Jurkat cell line (lymphocyte), and human umbilical vein endothelial cell (HUVEC, endothelial cell). Molecular targeting of P947 was confirmed by fluorimetry, ICP-MS, and in vitro MRI approaches. Potential application of P947 for detecting atherosclerotic plaques by in vivo MRI was tested in a rabbit model of atherosclerosis. In vitro, P947 displayed affinities for purified MMPs, ACE, ECE-1, NEP, APA, and APN in the micromolar range. Interestingly, MMPs, ACE, and APN exhibited higher activities in vulnerable plaques from human atherosclerotic carotid samples, as compared to stable plaques. ECE-1, NEP, and APA had either no activity or the same low activity in both vulnerable and stable plaques. P947 showed micromolar affinities for MMPs, ACE, and APN secreted by plaque samples. Moreover, P947 bound to THP-1 macrophages and THP-1 foam cells in a concentration-dependent manner and with a higher intensity than the control contrast agents DOTA-Gd or P1135 (DOTA-Gd coupled to a scrambled peptide). In THP-1 macrophages, P947 inhibited largely (70%) and almost completely (95%) MMP and APN activities, respectively, which strongly suggested an MMP- and APN-dependent binding of P947 to these cells. This enzyme-specific binding was confirmed with in vitro MRI. Indeed, the T1 value of THP-1 cells decreased from 2.094 s (macrophages w/o P947) to 2.004 s (macrophages with 1 mM of P947). In addition, the Gd content measured by ICP-MS was 11.01 ± 1.05 fg Gd/macrophage when cells were incubated in the presence of P947 and only 5.18 ± 0.43 fg Gd/macrophage with the control product P1135. The difference of Gd concentration between both contrast agents corresponded to a specific accumulation of 5.83 fg Gd/cell, which may be detected by MRI. MR imaging in the atherosclerosis rabbit model showed enhancement of the aortic wall after P947 injection with a significant increase of CNR values from 0.21 ± 0.02 (before injection) to 0.37 ± 0.07 (after injection), demonstrating the efficacy of the contrast agent to detect the atherosclerotic plaques in vivo. Taken together, these data suggest that P947 may be an interesting contrast agent for in vivo molecular MR imaging of MMPs, ACE, and APN activities present in vulnerable atherosclerotic plaques.

Relationship between vulnerability to reinforcing effects of morphine and activity of the endogenous cholecystokinin system in Lewis and Fischer rats.

A great number of studies have shown the presence of physiological interactions between brain neurotransmitter systems in behavioural responses. This is the case for opioid, cholecystokinin (CCK) and dopamine systems. However, so far the role that the CCK system may play in vulnerability to consumption of drugs of abuse is not clear. This was investigated in this study using Lewis rats that are more sensitive to the reinforcing properties of drugs of abuse than Fischer rats. The extraneuronal CCK(8) levels and brain CCK(2) receptors were found higher in Fischer than in Lewis rats in the nucleus accumbens, one of the most important structures involved in drug consumption. Moreover, pharmacological modulation of the CCK system by administration of a selective CCK(2) agonist blocked, in the conditioned place preference, the reinforcing effects of morphine in Lewis rats, whereas a selective CCK(2) antagonist revealed reinforcing effects of the alkaloid in Fischer rats. These results obtained following systemic administrations of the CCK ligands were confirmed following microinjection into the nucleus accumbens. Thus, a low level of CCK efflux in the nucleus accumbens could be one of the many factors involved in drug reinforcing effects, whereas a high level of CCK efflux could attenuate it.

Molecular mechanisms in Alzheimer's disease and related potential treatments such as structural target convergence of antibodies and simple organic molecules.

The amyloid cascade is the most frequently accepted hypothesis of Alzheimer's Disease (AD). According to this hypothesis, the formation of plaques precedes the appearance of fibrillary tangles. Therapeutic agents able to inhibit the formation of plaques are therefore considered as potential disease-modifying treatments (DMT) that could prevent or limit the progression of AD. Plaques are deposits formed by aggregates of amyloid-ß (Aß)-peptides. These peptides are metabolites of amyloid precursor protein (APP) first mediated by two enzymes: ß-secretase 1 (BACE1) and γ-secretase. Molecular identification of these two enzymes has stimulated the development of their inhibitors. The clinical testing of these two classes of molecules has not been successful to date. The oligomerization of Aß-peptides into plaques is now targeted by immunological approaches such as antibodies and vaccines. Structural consideration of the Aß-peptide sequence led to the launch of the antibody Aducanumab. Several other antibodies are in late clinical phases. Progress in the understanding of the effects of N-truncated Aß-peptides such as pE3-42, formed by the action of recently well characterized enzymes (aminopeptidase A, dipeptidylpeptidase-4 and glutaminyl cyclase) suggests that oligomerization can be limited either by enzyme inhibitors or antibody approaches. This strategy associating two structurally interconnected mechanisms is focused in this review.

Identification of an endothelin-converting enzyme-2-specific fluorigenic substrate and development of an in vitro and ex vivo enzymatic assay.

Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.

Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation.

Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)(n)(-)Nop-GGK-NH(2), X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.

Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250.

Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H(3)N(+)-CH(CH(3))-P(O)(OH)-CH(2)-CH(CH(2)Ph)-CONH-CH(CH(2)Ph)CO(2)(-), which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a K(i) of 1.5-2.2 nM and its three-dimensional structure in complex with E. coli APN showed its interaction with the S(1), S'(1) and S'(2) subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S'(2) subsite account for the 100-fold increase in affinity of PL250 compared with the dipeptide-derived inhibitor bestatin (K(i) = 4.1 x 10(-6) M). Accordingly, the removal of the C-terminal phenylalanine of PL250 resulted in a large decrease in affinity (K(i) = 2.17 x 10(-7) M). Furthermore, it was observed that the C-terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies.

Detection and quantification of botulinum neurotoxin type a by a novel rapid in vitro fluorimetric assay.

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle(202)]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH(2) acetylated at positions 156 to 203 [Nop(197), Pya(200), Nle(202)]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 x 10(6) M(-1) s(-1) and 8.85 x 10(6) M(-1) s(-1), respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.

Use of preproenkephalin knockout mice and selective inhibitors of enkephalinases to investigate the role of enkephalins in various behaviours.

RATIONALE: The most simple and efficient method to study the physiological role of enkephalins is to increase the lifetime of these endogenous opioid peptides by inhibiting their inactivating enzymes. Enkephalins are degraded by the concomitant action of two metallopeptidases: neutral endopeptidase (NEP, EC3.4.21.11) and aminopeptidase N (APN, EC3.4.11.2), both enzymes releasing inactive metabolites. OBJECTIVES: Potent dual inhibitors have been developed, such as RB101. However, NEP and APN have a broad specificity and can cleave various peptides in vitro. Therefore, it was essential to investigate the specific involvement of enkephalins in the various pharmacological responses induced by dual inhibitors. MATERIALS AND METHODS: We compared the pharmacological responses induced by RB101 in wild-type and preproenkephalin-deficient mice (Penk1-/-) using several behavioural assays. RESULTS: In all the tests used (hot plate test, force swim test, castor-oil-induced diarrhoea), RB101 induced strong effects in wild-type animals, whereas slight effects were observed in Penk1-/- animals. These residual effects are blocked by pre-administration of the opioid antagonist naloxone, supporting the involvement of the opioid receptors in the responses observed. CONCLUSIONS: The pharmacological effects induced by dual inhibitors acting on both NEP and APN are mainly due to the protection of the endogenous enkephalins at supraspinal and peripheral levels. It could be speculated that the residual effects observed in Penk1-/- mice after RB101 administration could be due to the direct action of other opioid peptides or through an indirect effect involving the protection of other peptide substrates of NEP or APN, as substance P or angiotensin.

Inhibition of osteosarcoma-induced thermal hyperalgesia in mice by the orally active dual enkephalinase inhibitor PL37. Potentiation by gabapentin.

We have previously shown that stimulation of peripheral opioid receptors by exogenous opiates counteracts the thermal hyperalgesia elicited by a tibial osteosarcoma due to intraosteal inoculation of NCTC 2472 cells to mice. Aiming to study whether pheripheral endogenous enkephalins could also counteract this painful symptom, we assayed in this model the effects of PL37, an orally active dual inhibitor of enkephalin inactivating enzymes. Oral administration of PL37 (25 mg/kg) completely supressed osteosarcoma-induced thermal hyperalgesia through the activation of micro-opioid receptors, since the administration of cyprodime (1 mg/kg) inhibited its antihyperalgesic effect. Neither naltrindole (0.1 mg/kg) nor nor-binaltorphimine (10 mg/kg) modified this PL37-induced antihyperalgesic effect. Moreover, the inhibition of the antihyperalgesic effect induced by PL37 after the administration of naloxone-methiodide (2 mg/kg), a non selective opioid antagonist that does not cross the blood-brain barrier, demonstrates the involvement of peripheral opioid receptors. In contrast, centrally mediated effects may be detected when assaying a higher dose of PL37 (50 mg/kg). Besides, the administration of gabapentin (6.25-25 mg/kg, i.p.) dose-dependently inhibited osteosarcoma-induced thermal hyperalgesia. Interestingly, the combined administration of subeffective doses of PL37 and gabapentin completely prevented this type of thermal hyperalgesia. An isobolographic analysis of this interaction demonstrated a synergistic interaction between both drugs.

Obligatory Metabolism of Angiotensin II to Angiotensin III for Zona Glomerulosa Cell-Mediated Relaxations of Bovine Adrenal Cortical Arteries.

Hyperaldosteronism is associated with hypertension, cardiac hypertrophy, and congestive heart failure. Steroidogenic factors facilitate aldosterone secretion by increasing adrenal blood flow. Angiotensin (Ang) II decreases adrenal vascular tone through release of zona glomerulosa (ZG) cell-derived vasodilatory eicosanoids. However, ZG cell-mediated relaxation of bovine adrenal cortical arteries to Ang II is not altered by angiotensin type 1 or 2 receptor antagonists. Because traditional Ang II receptors do not mediate these vasorelaxations to Ang II, we investigated the role of Ang II metabolites. Ang III was identified by liquid chromatography-mass spectrometry as the primary ZG cell metabolite of Ang II. Ang III stimulated ZG cell-mediated relaxation of adrenal arteries with greater potency than did Ang II. Furthermore, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by aminopeptidase inhibition, and Ang III-stimulated relaxations persisted. Ang IV had little effect compared with Ang II. Moreover, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by an Ang III antagonist but not by an Ang (1-7) antagonist. In contrast, Ang II and Ang III were equipotent in stimulating aldosterone secretion from ZG cells and were unaffected by aminopeptidase inhibition. Additionally, aspartyl and leucyl aminopeptidases, which convert Ang II to Ang III, are the primary peptidase expressed in ZG cells. This was confirmed by enzyme activity. These data indicate that intra-adrenal metabolism of Ang II to Ang III is required for ZG cell-mediated relaxations of adrenal arteries but not aldosterone secretion. These studies have defined an important role of Ang III in the adrenal gland.

Preproenkephalin knockout mice show no depression-related phenotype.

Clinical, preclinical, and pharmacological studies have suggested that decreased enkephalin tone is associated with depression-like symptoms and increase in enkephalin signaling could have a therapeutic value in the treatment of depression. In this study we demonstrate that, surprisingly, animals lacking enkephalin (preproenkephalin, Penk1(-/-)) showed no depression-related phenotype in the Porsolt forced swimming or tail suspension tests. Moreover, Penk1(-/-) mice had a lower frequency of depression-related behavior in stress-induced hypoactivity and ultrasonic vocalization models of depression, similar to animals treated with antidepressant drugs, although this effect was specific to the genetic background. In addition, there was no significant difference in the efficacy of antidepressant reference compounds in wild-type and knockout animals. Nialamide and amitriptyline were even slightly more effective in animals with genetic deletion of Penk1, whereas the minimal effective dose of imipramine and fluoxetine was the same in the two genotypes. The dual peptidase inhibitor RB-101 was also effective in Penk1(-/-) as well as in Penk1(-/-)/Pdyn(-/-) animals, although its efficacy was somewhat reduced compared with wild-type animals. This result was also surprising because the antidepressant effects of RB-101 were thought to be due to the elevation of enkephalin levels.

Evidence that creation of invasion sites determines the rate of strand transfer mediated by HIV-1 reverse transcriptase.

Strand transfer during reverse transcription can produce genetic recombination in human immunodeficiency virus type 1 (HIV-1) when two genomic RNAs, that are not identical, are co-packaged in the virus. Strand transfer was measured in vitro, in reactions involving primer switching from a donor to acceptor RNA template. The transfer product appeared with much slower kinetics than full-length synthesis on the donor template. The goal of this study was to learn more about the transfer mechanism by defining the steps that limit its rate. We previously proposed transfer to include the steps of acceptor invasion, hybrid propagation, terminus transfer, and re-initiation of synthesis on the acceptor template. Unexpectedly, with our templates increasing acceptor concentration increased the transfer efficiency but had no effect on the rate of transfer. Templates with a short region of homology limiting hybrid propagation exhibited a slow accumulation of transfer products, suggesting that for tested long homology templates hybrid propagation was not rate limiting. Substituting a DNA acceptor and adding Klenow polymerase accelerated re-initiation and extension exclusively on the DNA acceptor. This lead to a small rate increase due to faster extension on the acceptor, suggesting re-initiation of synthesis on the tested RNA acceptors was not rate limiting. A substrate was designed in which the 5' end of the primer was single stranded, and complimentary to the acceptor, i.e. having a pre-made invasion site. With this substrate, increasing concentrations of acceptor increased the rate of transfer. Together these data suggest that RNase H cleavage, and dissociation of RNA fragments creating an invasion site was rate limiting on most tested templates. When an accessible invasion site was present, acceptor interaction at that site influence the rate.